cc_byBerg, ChristophBusch, SelmaAlawiyah, Muthia DewiFinger, MauriceIhling, NinaPaquet-Durand, OlivierHitzmann, BerndBüchs, Jochen2024-09-032024-09-032023https://hohpublica.uni-hohenheim.de/handle/123456789/16187https://doi.org/10.1002/bit.28474Online fluorescence monitoring has become a key technology in modern bioprocess development, as it provides in‐depth process knowledge at comparably low costs. In particular, the technology is widely established for high‐throughput microbioreactor cultivation systems, due to its noninvasive character. For microtiter plates, previously also multi‐wavelength 2D fluorescence monitoring was developed. To overcome an observed limitation of fluorescence sensitivity, this study presents a modified spectroscopic setup, including a tunable emission monochromator. The new optical component enables the separation of the scattered and fluorescent light measurements, which allows for the adjustment of integration times of the charge‐coupled device detector. The resulting increased fluorescence sensitivity positively affected the performance of principal component analysis for spectral data of Escherichia coli batch cultivation experiments with varying sorbitol concentration supplementation. In direct comparison with spectral data recorded at short integration times, more biologically consistent signal dynamics were calculated. Furthermore, during partial least square regression for E. coli cultivation experiments with varying glucose concentrations, improved modeling performance was observed. Especially, for the growth‐uncoupled acetate concentration, a considerable improvement of the root‐mean‐square error from 0.25 to 0.17 g/L was achieved. In conclusion, the modified setup represents another important step in advancing 2D fluorescence monitoring in microtiter plates.eng2D fluorescence spectroscopyEscherichia coliHigh‐throughputMicrotiter plateMultivariate data analysisOnline monitoring660Article